Recombinant protein pharmaceutics have already provided unique therapies for several previously untreated diseases and numerous new protein drugs are being developed.
Proteins are usually administered parenterally, which can lead to a rapid elimination of the protein from the circulation. In order to maintain therapeutically effective blood levels, it is often necessary to administer large or frequent doses. The inconvenience and potential adverse side effects of this approach might be circumvented by employing systems that provide sustained or controlled delivery of the protein.
Sustained delivery systems can achieve more constant blood levels of protein therapeutics than those obtained with bolus doses, leading to improved drug efficacy and fewer adverse side effects. Those drug delivery systems include injectable oils, emulsions, suspensions, liposomes, microparticulates (microcapsules or microspheres), implants or gel systems.
Among gel systems used in drug delivery, poloxamer gels are used for their unique property as thermoset gel-forming materials in situ. Poloxamers are block copolymers of poly(ethylene oxide) and poly(propylene oxide), well-known as non-ionic surfactants that form aqueous gels which undergo transitions from a low to a high viscous state as a consequence of an increase in temperature, called “thermal gelation”.
In addition, Poloxamers possess good wetting, anti-foaming and solubilizing properties and are commonly used for pharmaceutical and medical purposes as drug delivery vehicles (Guzmán et al., 1992, International Journal of Pharmaceutics, 80, 119-127; Gander et al., 1986, Drug Dev. and Indust. Pharmacy, 12 (11-13), 1613-1623).
Poloxamers, referred by the trade name Pluronics® are tri-block copolymers having the following Formula (I):
wherein (a) and (c) are statistically equal, (b) is equal or higher than 15 and (a+c) form 20 to 90% of the mass of the molecule.
The two polyethylene oxide chains (PEO) are hydrophilic while the polypropylene chain (PPO) is hydrophobic, giving to the PEO-PPO-PEO block copolymers amphiphilic properties that can be modulated by varying the numbers of units (a) and (b).
Due to their amphiphilic nature, PEO-PPO-PEO block copolymers are able to self-aggregate to form a variety of associated structures such as micelles and liquid crystalline phases, as well as microemulsions.
Among Pluronics®, Poloxamer 407 (Lutrol® F127 or Pluronic® F127), a poloxamer of Formula (I) wherein (a)=(c)=99 and (b)=65 and Poloxamer 338 (Lutrol® F108 or Pluronic® F108), a poloxamer of Formula (I) wherein (a)=(c)=16 and (b)=46 are known for their thermal gelation properties of their aqueous solutions in the 20-35% concentration (Guzmán et al., 1992, above). Particularly, a 22-25% (w/w) Poloxamer 407 polymer solution is liquid at relatively low temperatures, i.e. 4-10° C., but rapidly forms a highly viscous, firm gel upon warming above a characteristic transition temperature, i.e. 18-20° C. These gels have been used for example for liquid hydrogel formulations for sub-cutaneous injections, topical applications, aerosols that form a gel as it warms to body temperature (Guzmán et al., 1992, above).
Poloxamer 407 gels have been found to enhance the stability of proteins loaded into the gel matrix (Stratton et al., 1997, Journal of Pharmaceutical sciences, 86, 9, 1006-1010) and have been used for various formulations including Lidocaine (Chen-Chow et al., 1981, International Journal of Pharmaceutics, 8, 89-99), Indomethacin (Miyazaki et al., 1986, Chem. Pharm. Bull. 34(4), 1801-1808) and IL-2 (Johnston et al., 1992, Pharmaceutical Research, 9(3), 425-434).
Interferons are cytokines, i.e. soluble proteins that transmit messages between cells and play an essential role in the immune system by helping to destroy microorganisms that cause infection and repairing any resulting damage. Interferons are naturally secreted by infected cells and were first identified in 1957. Their name is derived from the fact that they “interfere” with viral replication and production.
Interferons exhibit both antiviral and anti-proliferative activity. On the basis of biochemical and immunological properties, the naturally occurring human interferons are grouped into three major classes: interferon-alpha (leukocyte), interferon-beta (fibroblast) and interferon-gamma (immune). Alpha-interferon is currently approved in the United States and other countries for the treatment of hairy cell leukemia, venereal warts, Kaposi's Sarcoma (a cancer commonly afflicting patients suffering from Acquired Immune Deficiency Syndrome (AIDS)), and chronic non-A, non-B hepatitis.
Further, interferons (IFNs) are glycoproteins produced by the body in response to a viral infection. They inhibit the multiplication of viruses in protected cells. Consisting of a lower molecular weight protein, IFNs are remarkably non-specific in their action, i.e. IFN induced by one virus is effective against a broad range of other viruses. They are however species-specific, i.e. IFN produced by one species will only stimulate antiviral activity in cells of the same or a closely related species. IFNs were the first group of cytokines to be exploited for their potential anti-tumor and antiviral activities.
The three major IFNs are referred to as IFN-α, IFN-β and IFN-γ. Such main kinds of IFNs were initially classified according to their cells of origin (leukocyte, fibroblast or T cell). However, it became clear that several types might be produced by one cell. Hence leukocyte IFN is now called IFN-α, fibroblast IFN is IFN-β and T cell IFN is IFN-γ. There is also a fourth type of IFN, lymphoblastoid IFN, produced in the “Namalwa” cell line (derived from Burkitt's lymphoma), which seems to produce a mixture of both leukocyte and fibroblast IFN.
The interferon unit or international unit for interferon (U or IU, for international unit) has been reported as a measure of IFN activity defined as the amount necessary to protect 50% of the cells against viral damage. The assay that may be used to measure bioactivity is the cytopathic effect inhibition assay as described (Rubinstein, et al. 1981, J. Virol., 37, 755-758; Familletti et al., 1981, Methods in Enzymology, 78, Pestka Ed., Academic press, New York, 387-394). In this antiviral assay for interferon about 1 unit/ml of interferon is the quantity necessary to produce a cytopathic effect of 50%. The units are determined with respect to the international reference standard for Hu-IFN-beta provided by the National Institutes of Health (Pestka, 1986, Methods in Enzymology, 78, Pestka Ed., Academic press, New York 119, 14-23).
Every class of IFN contains several distinct types. IFN-β and IFN-γ are each the product of a single gene.
The proteins classified as IFNs-α are the most diverse group, containing about 15 types. There is a cluster of IFN-α genes on chromosome 9, containing at least 23 members, of which 15 are active and transcribed. Mature IFNs-α are not glycosylated.
IFNs-α and IFN-β are all the same length (165 or 166 amino acids) with similar biological activities. IFNs-γ are 146 amino acids in length, and resemble the α and β classes less closely. Only IFNs-γ can activate macrophages or induce the maturation of killer T cells. These new types of therapeutic agents can be sometimes called biologic response modifiers (BRMs), because they have an effect on the response of the organism to the tumor, affecting recognition via immunomodulation.
Human fibroblast interferon (IFN-β) has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is a polypeptide of about 20,000 Da induced by viruses and double-stranded RNAs. From the nucleotide sequence of the gene for fibroblast interferon, cloned by recombinant DNA technology, (Derynk et al., 1980, Nature, 285, 542-547) deduced the complete amino acid sequence of the protein. It is 166 amino acid long.
Shepard et al., 1981, Nature, 294, 563-565 described a mutation at base 842 (Cys→Tyr at position 141) that abolished its anti-viral activity, and a variant clone with a deletion of nucleotides 1119-1121.
Mark et al., 1984, Proc. Natl., Acad. Sci. U.S.A., 81(18), 5662-5666 inserted an artificial mutation by replacing base 469 (T) with (A) causing an amino acid switch from Cys→Ser at position 17. The resulting IFN-β was reported to be as active as the ‘native’ IFN-β and stable during long-term storage (−70° C.).
Rebif® (Serono—recombinant interferon-β), the latest development in interferon therapy for multiple sclerosis (MS) is interferon (IFN)-beta-1a produced from mammalian cell lines. Its recommended International Non-proprietary Name (INN) is “Interferon beta-1a”.
Various formulations of IFNs with copolymers have been developed in the past decades. Among them, IFN alpha injection formulations containing polyoxyethylene polyoxypropylene glycol (JP 2003 342193), cyclaradine-IFN alpha combined formulations (EP 0177153), kits for interferon alpha room-temperature Poloxamers gels for topical administration (U.S. Pat. No. 4,469,228), microparticle formulations of IFNβ (WO 01/58474), compositions comprising glycoproteins chemically coupled with polyoxyethylene-polyoxypropylene copolymer (EP 0098110) and IFNβ formulations for mucosal, especially intra-nasal, delivery (WO 2004/002404) have been described.
As with all protein-based pharmaceuticals, one major challenge in the use of an interferon as a therapeutic agent, is to maintain a therapeutically effective dose in the blood level for a certain time without increasing the injected dose and the potential associated side effects. Consequently, there is a need for IFN pharmaceutical compositions that sustain IFN plasma levels for a longer period of time than liquid formulations and/or that provide a higher plasma exposure of IFN, thereby maintaining or improving IFN biological activity.